Anti-fibrotic compositions comprising fendrr or fragments or variants thereof and methods of production and use thereof

ABSTRACT

Compositions containing long non-coding RNAs (IncRNA&#39;s) or fragments or variants thereof are disclosed. Also disclosed are compositions containing vectors that include or encode the IncRNA&#39;s or fragments/variants thereof. Further disclosed are methods of producing and using these compositions containing or encoding the IncRNA&#39;s.

CROSS REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCESTATEMENT

The subject application claims benefit under 35 USC § 119(e) ofprovisional application U.S. Ser. No. 62/926,797, filed Oct. 28, 2019.The entire contents of the above-referenced patent(s)/patentapplication(s) are hereby expressly incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. Government support under Grant Nos.R01HL135152, R01HL116876, and P20GM103648 awarded by the NationalInstitutes of Health. The Government has certain rights in thisinvention.

BACKGROUND

Idiopathic Pulmonary Fibrosis (IPF) is a chronic and lethal fibroticlung disease characterized by scarring of lung tissues and worseninglung function. Historically, lung tissues from IPF patients show similarcharacteristics as usual interstitial pneumonia (UIP). The diseaseprimarily occurs in individuals between the ages of 50 and 70 and morefrequently occurs in men. The course of IPF is difficult to predict,with a median survival time of 3 to 5 years after diagnosis; there iscurrently no effective therapy.

The human genome generates more non-coding RNAs (ncRNAs) thanprotein-coding RNA sequences. Long non-coding RNAs (IncRNAs) are ncRNAsthat are typically longer than 200 nt and are transcribed from DNA thatwas once thought to be “junk.” These RNAs are larger than small ncRNAssuch as microRNAs, Piwi-interacting RNAs, siRNAs, and small nucleolarRNAs, which are typically about 20-180 nt in length. Current estimatessuggest that about 20,000 distinct IncRNAs are present in humans.

Most ncRNAs, including long non-coding RNAs (IncRNAs), are synthesizedby polymerase II. IncRNAs are mRNA-like transcripts, but are non-proteinencoding RNA molecules that are more than 200 bp long. IncRNAs areprocessed by capping, splicing, and polyadenylation, which is similar tothe process of protein-coding genes. Only a small number of IncRNAs havebeen annotated functionally. IncRNAs are located in the nucleus,cytoplasm, and mitochondria, where they participate in various molecularfunctions, such as chromatin remodeling, transcriptional regulation, RNAsplicing, RNA stability, and translation control. IncRNAs are importantin controlling critical physiological functions, including geneimprinting, cell proliferation, differentiation, apoptosis, autophagy,migration, immune responses, and chromosome structure. Aberrantexpression of IncRNAs has been associated with a broad range of humandiseases, including cardiovascular, neurodegenerative, metabolic, andlung diseases, as well as tumors and infections.

Fetal-lethal noncoding developmental regulatory RNA (FENDRR) is a IncRNAthat is transcribed bidirectionally with FOXF1 on its opposite strand.FENDRR binds to polycomb repressive complex 2 (PRC2) and/or TrxG/MLLcomplexes to epigenetically regulate the expression of its target genes.Murine Fendrr is essential for normal development of the lungs, heart,and body wall. LacZ reporter profiling has shown that Fendrr is highlyexpressed in embryonic and adult lung tissue. Fendrr homozygotes areembryonic-lethal due to defective structural maturation of the lungs.Genomic deletion within the FENDRR gene was found in a human fatal lungdevelopment disorder, alveolar capillary dysplasia with misalignment ofpulmonary veins (ACD/MPV). FENDRR has been linked to other humandiseases, such as gastric cancer.

Therefore, there is a need in the art for new and improved compositionsthat contain or encode IncRNAs, such as (but not limited to), FENDRR.There is also a need in the art for methods of inhibiting activation offibroblasts and treating or reducing the occurrence of pulmonaryfibrosis. It is to such compositions and methods that the presentdisclosure is directed.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. FENDRR was down-regulated in fibrotic lungs and fibroblasts.Panel (A): Next-generation RNA sequencing analysis showing thedown-regulation of FENDRR in IPF lungs in two published datasets. Panel(B): Real-time PCR showing FENDRR down-regulation in LTRC IPF lungs. n=7for Control, n=27 for IPF. The primers detecting human FENDRR transcriptvariant 2 and 3 were used. Panel (C): Real-time PCR showing FENDRRdown-regulation in fibrotic mouse lungs (n=3). Panel (D): Real-time PCRshowing FENDRR expression in primary fibroblasts and alveolar epithelialtype I and type II cells (AEC I and AEC II) isolated from saline (Sal)-and bleomycin (Bleo)-treated mice (n=3). The expression levels werenormalized to GAPDH. Data are expressed as the percent of the fibroblastcontrol group. The primers detecting mouse Fendrr transcript variants 1and 2 were used for C and D. Panel (E) shows the copy number of humanFENDRR transcript variants in the fibroblasts, as determined by absolutequantitative Real-time PCR (n=3). Panel (F) shows the copy number ofhuman FENDRR variants in LTRC IPF lungs, as determined by dropletdigital PCR. n=7 for Control, n=27 for IPF. Panel (G) shows the copynumber of mouse Fendrr variants in the lungs of control andbleomycin-treated mice, as determined by droplet digital PCR (n=3). Theresults are presented as the means±SEM. Student's t-test for A, B, and Cand ANOVA, followed by Tukey's HSD test for D, F, and G.

FIG. 2. Gene structure of FENDRR and primer locations used fordetermining the expression of FENDRR transcript variants. red color:forward primer binding site; and green color: reverse primer bindingsite. purple color: common primers for human variants 2, 3 and mousevariants 1, 2.

FIG. 3. TGFβ1-SMAD3 signaling inhibits FENDRR expression in lungfibroblasts. Panel (A): Real-time PCR showing FENDRR down-regulation inHPF fibroblasts treated with TGFβ1 (5 ng/ml) for 48 hours (n=3). Panel(B) shows the copy number of FENDRR variants in the control andTGFβ1-treated HPF fibroblasts, as determined by droplet digital PCR(n=4). Panel (C): Dual luciferase reporter assay showing the inhibitionof human FENDRR promoter activity by TGFβ1 in HPF and HFL1 cells.Firefly luciferase activity was normalized to Renilla luciferaseactivity. Fold change was calculated relative to control (n=3). Panel(D): Western blot analysis showing knockdown of SMAD2/3 expression inLL29 cells by shRNA. Panel (E): Real-time PCR showing that FENDRRexpression in LL29 cells was inhibited by TGFβ1 and rescued by SMAD3knockdown (n=3). The results are presented as the means±SEM. Student'st-test for A and ANOVA, followed by Tukey's HSD test for B, C and E.

FIG. 4. Overexpression of FENDRR inhibits TGFβ1-induced fibroblastactivation. LL29 cells stably expressing FENDRR transcript variant 3 ora control (VC) were treated with 5 ng/ml of TGFβ1 for 48 hours. Panel(A) shows FENDRR expression (n=3). Panel (B) shows that FENDRRoverexpression suppressed TGF-β1-induced mRNA expression of α-SMA,COL1A1, and COL3A1. FENDRR and mRNA expression were determined byreal-time PCR and normalized to GAPDH (n=3). Panel (C) shows Westernblot analysis demonstrating the suppression of TGFβ1-induced α-SMA,COL1A1, and COL3A1 protein expression levels by FENDRR overexpression.Panel (D) shows FENDRR reduced TGFβ1-induced collagen gel contraction.n=4. Panel (E) shows immunostaining demontrating the inhibition of fiberformation by FENDRR using anti-α-SMA antibodies and Alexa 546-conjugatedsecond antibodies. Scale bar: 50 μm. Panels (F, G): HPF fibroblasts wereinfected with a lentiviral FENDRR shRNA or its control (shCON) (MOI=50)for two days, and the cells were collected for analysis. Real-time PCRand western blotting showing that the knockdown of FENDRR enhanced mRNAexpression of α-SMA, COL1A1, and COL3A1 and protein expression of α-SMAand COL1A1. The results are expressed as fold changes relative to shCON.n=3. The results are presented as the means±SEM. Student's t-test for A,F, ANOVA, followed by Tukey's HSD test for B and ANOVA, followed byuncorrected Fisher's LSD test for D.

FIG. 5. IRP1 is an interacting partner of FENDRR. Panel (A) shows thatFENDRR is preferentially localized in the cytoplasm in fibroblasts. TheRNA levels in cytoplasmic and nuclear fractions of LL29 and HPFfibroblasts were determined by real-time PCR and calculated with theequation 2^(−Ct). GAPDH, ACTB and U2snRNA were used as controls forcytoplasmic and nuclear RNA, respectively (n=3). Panel (B) shows a RIPassay demonstrating the interaction of FENDRR and IRP1 in LL29fibroblasts (n=3). Panel (C) shows mapping of the binding region ofFENDRR with IRP1 by CLIP-qPCR analysis (n=3). Data are presented as themeans±SEM. Student's t-test for B.

FIG. 6. Predicted RNA structure of FENDRR transcript variant 3. Blackcolor: start and ending position. Red color: start and end positions ofthe IRP1 binding region.

FIG. 7. FENDRR controls iron metabolism by interacting with IRP1. Panel(A) shows that FENDRR overexpression decreased iron levels in LL29fibroblasts, as determined with an Iron Assay Kit. n=4. Panel (B) showsthat FENDRR overexpression inhibited aconitase activity in LL29fibroblasts, as measured using an Aconitase Enzyme Activity MicroplateAssay Kit. n=3. Panel (C) shows real-time PCR demonstrating thesuppression of the expression of TFRC mRNA with FENDRR overexpression.n=4. Panel (D) shows that iron levels were increased in primaryfibroblasts isolated from the lungs of bleomycin (Bleo)-treated micecompared to that from saline (Sal)-treated mice (n=3). Panel (E): LL29cells stably expressing FENDRR or a control (VC) were infected withpooled three lentiviral IRP1 shRNAs or a control (shCON) (MOI=50) fortwo days, and the cells were then cultured in the serum-free RPMI-1640medium (iron-free medium) or the complete RPMI-1640 medium containing 10μM ferric ammonium citrate (iron-supplemented medium) for another twodays. Western blotting shows the knockdown of IRP1 expression. IRP1knockdown rescued the FENDRR-mediated decrease in cellular iron levelsin iron-free medium (n=6). Fold changes were calculated based on the VCand shCON control in the iron-free medium. Panels (F, G, H): LL29 lungfibroblasts were treated with 5 μM desferrioxamine (DFO) and with orwithout TGFβ1 (5 ng/ml) for 48 hours. The mRNA expression levels ofα-SMA, COL1A1 and COL3A1 were determined by real-time PCR (n=3). Dataare presented as the means±SEM. Student's t-test for A, B, C and D,ANOVA, followed by uncorrected Fisher's LSD test for E, and ANOVA,followed by Tukey's HSD for F, G, and H.

FIG. 8. miR-214 binding sites in human FENDRR transcript variant 3.

FIG. 9. FENDRR sponges miR-214. Panel (A) shows a luciferase assayshowing that FENDRR overexpression increased the activity of miR-214sensor in LL29 cells (n=4). Panel (B) shows a luciferase assay showingthat FENDRR increased miR-214 sensor activity by competing with miR-214in HEK293T cells. The effects of FENDRR on a miR-214 sensor werereversed by increasing the miR-214 level. HEK293T cells wereco-transfected with 5 ng miR-214 sensor and 20, 40, or 60 ngmiR-214/Control and 150 ng FENDRR-overexpression vector/control.Luciferase activities were determined 48 hours after transfection (n=4).Panels (C, D) show Real-time PCR and western blot analysis demonstratingincreases in TGF-β1-induced COL1A1 mRNA and protein levels by miR-214overexpression. LL29 cells were treated with a lentiviral miR-214 or thevirus control (VC) at an MOI of 50 for 48 hours. Then, the cells werestimulated with 5 ng/ml of TGF-β1 for 48 hours (n=4). Panel (E) showsReal-time PCR demonstrating the expression of primary miR-214(pri-miR214) and mature miR-214 in LL29 cells treated with DFO (n=3).Data are presented as the means±SEM. ANOVA, followed by uncorrectedFisher's LSD test for A, ANOVA, followed by Tukey's HSD test for B, Cand student's t-test for E.

FIG. 10. FENDRR overexpression attenuates bleomycin-induced mouse lungfibrosis. On day 0, mice were infected with AdFENDRR or AdCON (5×10⁹ IU)through nasal instillation. On day 1, saline (Sal) or bleomycin (Bleo)(1 U/kg BW) was delivered into the mouse lungs through nasalinstillation. At D14, the mice were subjected to an analysis ofrespiratory mechanics by Flexivent and then sacrificed. The left lungswere collected for RNA and protein analysis, and the right lungs werefixed for histological analysis. Panel (A) shows Real-time PCRdemonstrating FENDRR overexpression in mouse lungs. Primers detectinghuman FENDRR transcript variants 2 and 3 and murine Fendrr transcriptvariants 1 and 2 were used. Sal+AdCON (n=5), Sal+AdFENDRR (n=7),Bleo+AdCON (n=6), Bleo+AdFENDRR (n=8). Panel (B) shows H&E stainingdemonstrating fibrotic changes in mouse lungs induced by bleomycin.FENDRR attenuated the fibrotic changes in bleomycin-treated mouse lungs.Scale bar: 100 μm. Panel (C) shows Ashcroft score demonstrating thatFENDRR lung transfer attenuated bleomycin-induced mouse pulmonaryfibrosis. Sal+AdCON (n=15), Sal+AdFENDRR (n=20), Bleo+AdCON (n=21),Bleo+AdFENDRR (n=24). Panel (D) shows a Hydroxyproline assay. Sal+AdCON(n=5), Sal+AdFENDRR (n=7), Bleo+AdCON (n=6), Bleo+AdFENDRR (n=7). Panels(E, F) show Real-time PCR analysis demonstrating that FENDRR inhibitedbleomycin-induced Col1a1 and Col3a1 mRNA expression levels in mouselungs. Sal+AdCON (n=7), Sal+AdFENDRR (n=12), Bleo+AdCON (n=14),Bleo+AdFENDRR (n=16). Panels (G, H) show Western blotting demonstratingthat FENDRR inhibited bleomycin-induced COL1A1 protein expression inmouse lungs. Sal+AdCON (n=6), Sal+AdFENDRR (n=6), Bleo+AdCON (n=6),Bleo+AdFENDRR (n=6). Panel (I) shows that FENDRR improved respiratoryfunction in bleomycin-treated mouse by Flexi-vent analysis. Elastance(Ers) was measured in a single-compartment model. Sal+AdCON (n=6),Sal+AdFENDRR (n=7), Bleo+AdCON (n=6), Bleo+AdFENDRR (n=7). The resultsare presented as the means±SEM. ANOVA, followed by Tukey's HSD test forA, C, E, F, H and ANOVA, followed by uncorrected Fisher's LSD test forD, I.

FIG. 11. Schematic representation of one, non-limiting mechanistic modelfor the regulation of FENDRR in idiopathic pulmonary fibrosis.

DETAILED DESCRIPTION

Before explaining at least one embodiment of the inventive concept(s) indetail by way of exemplary language and results, it is to be understoodthat the inventive concept(s) is not limited in its application to thedetails of construction and the arrangement of the components set forthin the following description. The inventive concept(s) is capable ofother embodiments or of being practiced or carried out in various ways.As such, the language used herein is intended to be given the broadestpossible scope and meaning; and the embodiments are meant to beexemplary—not exhaustive. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

Unless otherwise defined herein, scientific and technical terms used inconnection with the presently disclosed inventive concept(s) shall havethe meanings that are commonly understood by those of ordinary skill inthe art. Further, unless otherwise required by context, singular termsshall include pluralities and plural terms shall include the singular.The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses and chemical analyses.

All patents, published patent applications, and non-patent publicationsmentioned in the specification are indicative of the level of skill ofthose skilled in the art to which this presently disclosed inventiveconcept(s) pertains. All patents, published patent applications, andnon-patent publications referenced in any portion of this applicationare herein expressly incorporated by reference in their entirety to thesame extent as if each individual patent or publication was specificallyand individually indicated to be incorporated by reference.

All of the compositions and/or methods disclosed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. While the compositions and methods of the inventiveconcept(s) have been described in terms of particular embodiments, itwill be apparent to those of skill in the art that variations may beapplied to the compositions and/or methods and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit, and scope of the inventive concept(s). All suchsimilar substitutions and modifications apparent to those skilled in theart are deemed to be within the spirit, scope, and concept of theinventive concept(s) as defined by the appended claims.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The use of the term “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” As such, the terms “a,” “an,” and “the”include plural referents unless the context clearly indicates otherwise.Thus, for example, reference to “a compound” may refer to one or morecompounds, two or more compounds, three or more compounds, four or morecompounds, or greater numbers of compounds. The term “plurality” refersto “two or more.”

The use of the term “at least one” will be understood to include one aswell as any quantity more than one, including but not limited to, 2, 3,4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term “at least one” mayextend up to 100 or 1000 or more, depending on the term to which it isattached; in addition, the quantities of 100/1000 are not to beconsidered limiting, as higher limits may also produce satisfactoryresults. In addition, the use of the term “at least one of X, Y, and Z”will be understood to include X alone, Y alone, and Z alone, as well asany combination of X, Y, and Z. The use of ordinal number terminology(i.e., “first,” “second,” “third,” “fourth,” etc.) is solely for thepurpose of differentiating between two or more items and is not meant toimply any sequence or order or importance to one item over another orany order of addition, for example.

The use of the term “or” in the claims is used to mean an inclusive“and/or” unless explicitly indicated to refer to alternatives only orunless the alternatives are mutually exclusive. For example, a condition“A or B” is satisfied by any of the following: A is true (or present)and B is false (or not present), A is false (or not present) and B istrue (or present), and both A and B are true (or present).

As used herein, any reference to “one embodiment,” “an embodiment,”“some embodiments,” “one example,” “for example,” or “an example” meansthat a particular element, feature, structure, or characteristicdescribed in connection with the embodiment is included in at least oneembodiment. The appearance of the phrase “in some embodiments” or “oneexample” in various places in the specification is not necessarily allreferring to the same embodiment, for example. Further, all referencesto one or more embodiments or examples are to be construed asnon-limiting to the claims.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for acomposition/apparatus/device, the method being employed to determine thevalue, or the variation that exists among the study subjects. Forexample, but not by way of limitation, when the term “about” isutilized, the designated value may vary by plus or minus twenty percent,or fifteen percent, or twelve percent, or eleven percent, or tenpercent, or nine percent, or eight percent, or seven percent, or sixpercent, or five percent, or four percent, or three percent, or twopercent, or one percent from the specified value, as such variations areappropriate to perform the disclosed methods and as understood bypersons having ordinary skill in the art.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”), or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, the term “substantially” means that the subsequentlydescribed event or circumstance completely occurs or that thesubsequently described event or circumstance occurs to a great extent ordegree. For example, when associated with a particular event orcircumstance, the term “substantially” means that the subsequentlydescribed event or circumstance occurs at least 80% of the time, or atleast 85% of the time, or at least 90% of the time, or at least 95% ofthe time. For example, the term “substantially adjacent” may mean thattwo items are 100% adjacent to one another, or that the two items arewithin close proximity to one another but not 100% adjacent to oneanother, or that a portion of one of the two items is not 100% adjacentto the other item but is within close proximity to the other item.

The term “polypeptide” as used herein will be understood to refer to apolymer of amino acids. The polymer may include d-, I-, or artificialvariants of amino acids. In addition, the term “polypeptide” will beunderstood to include peptides, proteins, and glycoproteins.

The term “polynucleotide” as used herein will be understood to refer toa polymer of two or more nucleotides. Nucleotides, as used herein, willbe understood to include deoxyribose nucleotides and/or ribosenucleotides, as well as artificial variants thereof. The termpolynucleotide also includes single-stranded and double-strandedmolecules.

The terms “analog” or “variant” as used herein will be understood torefer to a variation of the normal or standard form or the wild-typeform of molecules. For polypeptides or polynucleotides, an analog may bea variant (polymorphism), a mutant, and/or a naturally or artificiallychemically modified version of the wild-type polynucleotide (includingcombinations of the above). Such analogs may have higher, full,intermediate, or lower activity than the normal form of the molecule, orno activity at all. Alternatively and/or in addition thereto, for achemical, an analog may be any structure that has the desiredfunctionalities (including alterations or substitutions in the coremoiety), even if comprised of different atoms or isomeric arrangements.

As used herein, the phrases “associated with” and “coupled to” includeboth direct association/binding of two moieties to one another as wellas indirect association/binding of two moieties to one another.Non-limiting examples of associations/couplings include covalent bindingof one moiety to another moiety either by a direct bond or through aspacer group, non-covalent binding of one moiety to another moietyeither directly or by means of specific binding pair members bound tothe moieties, incorporation of one moiety into another moiety such as bydissolving one moiety in another moiety or by synthesis, and coating onemoiety on another moiety, for example.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%, 95%, and 99%. Most preferably, the object speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

The term “pharmaceutically acceptable” refers to compounds,compositions, and/or dosage forms which are, with the scope of soundmedical judgment, suitable for administration to humans and/or animalswithout undue adverse side effects such as (but not limited to)toxicity, irritation, and/or allergic response commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition, excipient, orvehicle, such as a liquid or solid filler, diluent, excipient, orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. The term “pharmaceutically-acceptablecarrier” refers to any carrier, vehicle, excipient, and/or diluent knownin the art or otherwise contemplated herein that may improve solubility,deliverability, dispersion, stability, and/or conformational integrityof the compositions disclosed herein.

The term “patient” as used herein includes human and veterinarysubjects. “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including (but not limited to) humans, domesticand farm animals, nonhuman primates, and any other animal that hasmammary tissue.

The term “child” is meant to refer to a human individual who would berecognized by one of skill in the art as an infant, toddler, etc., or anindividual less than about 18 years of age, usually less than about 16years of age, usually less than about 14 years of age, or even less(e.g., from newborn to about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, about 11, or about 12 years ofage). The term “elderly” generally refers to a human individual whoseage is greater than about 50 years of age, usually greater than about 55years of age, frequently greater than about 60 years of age or more(e.g., about 65 years of age and upwards).

The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude, but are not limited to, individuals already having a particularcondition/disease/infection as well as individuals who are at risk ofacquiring a particular condition/disease/infection (e.g., those needingprophylactic/preventative measures). The term “treating” refers toadministering an agent to a patient for therapeutic and/orprophylactic/preventative purposes.

A “therapeutic composition” or “pharmaceutical composition” refers to anagent that may be administered in vivo to bring about a therapeuticand/or prophylactic/preventative effect.

Administering a therapeutically effective amount or prophylacticallyeffective amount is intended to provide a therapeutic benefit in thetreatment, prevention, and/or management of a disease, condition, and/orinfection. The specific amount that is therapeutically effective can bereadily determined by the ordinary medical practitioner, and can varydepending on factors known in the art, such as (but not limited to) thetype of condition/disease/infection, the patient's history and age, thestage of the condition/disease/infection, and the co-administration ofother agents.

The term “effective amount” refers to an amount of a biologically activemolecule or conjugate or derivative thereof sufficient to exhibit adetectable therapeutic effect without undue adverse side effects (suchas (but not limited to) toxicity, irritation, and allergic response)commensurate with a reasonable benefit/risk ratio when used in themanner of the inventive concept(s). The therapeutic effect may include,for example but not by way of limitation, preventing, inhibiting, orreducing the occurrence of pulmonary fibrosis. The effective amount fora subject will depend upon the type of subject, the subject's size andhealth, the nature and severity of the condition/disease/infection to betreated, the method of administration, the duration of treatment, thenature of concurrent therapy (if any), the specific formulationsemployed, and the like. Thus, it is not possible to specify an exacteffective amount in advance. However, the effective amount for a givensituation can be determined by one of ordinary skill in the art usingroutine experimentation based on the information provided herein.

As used herein, the term “concurrent therapy” is used interchangeablywith the terms “combination therapy” and “adjunct therapy,” and will beunderstood to mean that the patient in need of treatment is treated orgiven another drug for the condition/disease/infection in conjunctionwith the pharmaceutical compositions of the present disclosure. Thisconcurrent therapy can be sequential therapy, where the patient istreated first with one pharmaceutical composition and then the otherpharmaceutical composition, or the two pharmaceutical compositions aregiven simultaneously.

The terms “administration” and “administering,” as used herein, will beunderstood to include all routes of administration known in the art,including but not limited to, oral, topical, transdermal, parenteral,subcutaneous, intranasal, intratracheal, intrabronchial, mucosal,intramuscular, intraperitoneal, intravitreal, and intravenous routes,and including both local and systemic applications. In addition, thecompositions of the present disclosure (and/or the methods ofadministration of same) may be designed to provide delayed, controlled,or sustained release using formulation techniques which are well knownin the art.

Turning now to the inventive concept(s), certain non-limitingembodiments thereof are directed to an anti-fibrotic composition thatcomprises an isolated and/or purified Fetal-lethal noncodingdevelopmental regulatory RNA (FENDRR) IncRNA or a fragment or variantthereof and a pharmaceutically-acceptable carrier. In certainnon-limiting embodiments, the FENDRR IncRNA or fragment/variant thereofis encoded by a sequence comprising at least about 100 contiguousnucleotides of at least one of SEQ ID NOS:1-3 and 133-134, or a sequencethat differs from at least one of SEQ ID NOS:1-3 and 133-134 by lessthan about 30 amino acids. SEQ ID NO:1 represents Human FENDRRtranscript variant 1 (GenBank Accession No. NR_036444), SEQ ID NO:2 isHuman FENDRR transcript variant 2 (GenBank Accession No. NR_033925), andSEQ ID NO:3 represents a novel Human FENDRR transcript variant 3disclosed herein (GenBank Accession No. MK522493.1). SEQ ID NO:133represents mouse FENDRR transcript variant 1 (GenBank Accession No.NR_130109), while SEQ ID NO:134 represents mouse FENDRR transcriptvariant 2 (GenBank Accession No. NR045471).

The fragment/variant of FENDRR IncRNA may be at least about 25contiguous nucleotides long, such as (but not limited to) at least about50, at least about 60, at least about 70, at least about 80, at leastabout 90, at least about 100, at least about 110, at least about 120, atleast about 130, at least about 140, at least about 150, at least about160, at least about 170, at least about 180, at least about 190, atleast about 200, at least about 210, at least about 220, at least about230, at least about 240, at least about 250, at least about 260, atleast about 270, at least about 280, at least about 290, at least about300, at least about 310, at least about 320, at least about 330, atleast about 340, at least about 350, at least about 360, at least about370, at least about 380, at least about 390, at least about 400, atleast about 425, at least about 450, at least about 475, at least about500, at least about 525, at least about 550, at least about 575, atleast about 600, at least about 650, at least about 700, at least about750, at least about 800, at least about 850, at least about 900, atleast about 950, at least about 1000, at least about 1100, at leastabout 1200, at least about 1300, at least about 1400, at least about1500, at least about 1600, at least about 1700, at least about 1800, atleast about 1900, at least about 2000, at least about 2100, at leastabout 2200, at least about 2300, at least about 2400, at least about2500, at least about 2600, at least about 2700, at least about 2800, atleast about 2900, at least about 3000, at least about 3100, or at leastabout 3200 nucleotides long, or the like. The scope of the presentdisclosure also includes fragments/variants of FENDRR IncRNA that have alength within a range of two of the above values (i.e., a range of fromabout 50 to about 500 nucleotides long, a range of from about 100 toabout 150 nucleotides long, etc.), as well as a length that fallsbetween two of any of the above values (i.e., at least about 725, atleast about 1450, etc.).

In a particular (but non-limiting) embodiment, the fragment or variantof FENDRR IncRNA may comprise any of the regions outlined in Table 4,such as, but not limited to the region between 1419-1549. However, itwill be understood that these “regions” are for purposes of exampleonly, and the fragment or variant may include additional sequence on oneor both sides thereof.

The fragment or variant of FENDRR IncRNA may be encoded by a sequencethat differs from at least one of SEQ ID NOS:1-3 and 133-134 by lessthan about 30 amino acids, such as, but not limited to, less than about29 amino acids, less than about 28 amino acids, less than about 27 aminoacids, less than about 26 amino acids, less than about 25 amino acids,less than about 24 amino acids, less than about 23 amino acids, lessthan about 22 amino acids, less than about 21 amino acids, less thanabout 20 amino acids, less than about 19 amino acids, less than about 18amino acids, less than about 17 amino acids, less than about 16 aminoacids, less than about 15 amino acids, less than about 14 amino acids,less than about 13 amino acids, less than about 12 amino acids, lessthan about 11 amino acids, less than about 10 amino acids, less thanabout 9 amino acids, less than about 8 amino acids, less than about 7amino acids, less than about 6 amino acids, less than about 5 aminoacids, and the like.

The FENDRR IncRNA or fragment or variant thereof may be modified and/orencapsulated so as to improve the stability thereof. Any nucleotidemodifications or encapsulation methods known in the art or otherwisecontemplated herein may be utilized in accordance with the presentdisclosure. For example (but not by way of limitation), the FENDRRIncRNA or fragment or variant thereof may be modified by one of moremodifications selected from a PS backbone modification, 2′-OMe, 2′-F,2′MOE, a sugar substitution, LNA, and/or L-RNA modification.

In addition (but not by way of limitation), the FENDRR IncRNA orfragment/variant thereof may be encapsulated in a delivery vehicle.Non-limiting examples of delivery vehicles that may be utilized inaccordance with the present disclosure include a liposome, a lipoplex, amicrovesicle, an exosome, a lipidoid nanoparticle, a polymericnanoparticle, an inorganic nanoparticle, and a stable nucleic acidparticle (SNALP), and the like, as well as variations, derivatives, andcombinations thereof.

Certain non-limiting embodiments of the present disclosure are directedto a pharmaceutical composition that comprises a vector comprising asequence encoding at least about 100 contiguous nucleotides of aFetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNA or afragment or variant thereof and a pharmaceutically-acceptable carrier.

Any sequences encoding FENDRR IncRNAs or fragments/variants thereof thatare known in the art or otherwise contemplated herein may be utilized inaccordance with the present disclosure. In certain non-limitingembodiments, the sequence comprises at least about 100 contiguousnucleotides of at least one of SEQ ID NOS:1-3 and 133-134, or a sequencethat differs from at least one of SEQ ID NOS:1-3 and 133-134 by lessthan about 30 amino acids.

Any vectors known in the art or otherwise contemplated herein may beutilized in accordance with the present disclosure, so long as thevector allows for expression of the FENDRR IncRNA or fragment/variantthereof. Non-limiting examples of vectors that can be utilized inaccordance with the present disclosure include an adenoviral vector, anadeno-associated viral (AAV) vector, an alpha viral vector, a herpesviral vector, a lentiviral vector, a measles viral vector, a pox viralvector, a phage vector, a retroviral vector, and the like.

The vector can include any other components/elements that aid the vectorin functioning in accordance with the present disclosure. For example(but not by way of limitation), the vector may further include anexpression control sequence to which the FENDRR sequence is operablylinked.

Any pharmaceutically-acceptable carriers known in the art or otherwisecontemplated herein may be utilized in accordance with the presentdisclosure. Non-limiting examples include a pharmaceutically acceptablesolvent, suspending agent, or vehicle that aid in delivery of thecompositions of the present disclosure to the human or animal. Thecarrier may be liquid or solid and is selected with the planned mannerof administration in mind. Examples of pharmaceutically acceptablecarriers that may be utilized in accordance with the present disclosureinclude, but are not limited to, PEG, liposomes, ethanol, DMSO, aqueousbuffers, oils, DPPC, lipids, other biologically-active molecules,vaccine-adjuvants, and combinations thereof. In addition, in certainparticular (but non-limiting) examples, pharmaceutically-acceptablecarriers can also contain a physiologically acceptable compound thatacts to stabilize the compound and/or increase or decrease theabsorption or clearance rates of the pharmaceutical compositions.Physiologically acceptable compounds can include (for example, but notby way of limitation) carbohydrates, such as glucose, sucrose, ordextrans; antioxidants, such as ascorbic acid or glutathione; chelatingagents; low molecular weight proteins; detergents; liposomal carriers;and/or excipients or other stabilizers and/or buffers. Otherphysiologically acceptable compounds include (for example, but not byway of limitation) wetting agents, emulsifying agents, dispersingagents, and/or preservatives.

The compositions of the present disclosure may be provided in any formand any formulation that allows the compositions to function inaccordance with the present disclosure. For example (but not by way oflimitation), the compositions may be in solid (such as, but not limitedto, tablets, powders, and dry powder inhalers), liquid (such as, but notlimited to, emulsions, microemulsions, solutions, suspensions, syrups,and elixirs), or gels, or sprays, mists, or aerosols.

Certain non-limiting embodiments of the present disclosure are directedto a method of inhibiting activation of lung fibroblasts. In the method,the lung fibroblasts are contacted with any of the compositionsdisclosed or otherwise contemplated herein. For example (but not by wayof limitation), the composition may be selected from: (i) an isolatedand/or purified Fetal-lethal noncoding developmental regulatory RNA(FENDRR) IncRNA or a fragment or variant thereof (as described in detailherein above or otherwise contemplated herein); and/or (ii) a vectorcomprising a sequence encoding at least about 100 contiguous nucleotidesof a Fetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNAor a fragment or variant thereof (as described in detail herein above orotherwise contemplated herein). In particular (but not by way oflimitation): in (i), the FENDRR IncRNA is encoded by a sequencecomprising at least about 100 contiguous nucleotides of at least one ofSEQ ID NOS:1-3 and 133-134, or a sequence that differs from at least oneof SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids; and in(ii), the sequence comprises at least about 100 contiguous nucleotidesof at least one of SEQ ID NOS:1-3 and 133-134, or a sequence thatdiffers from at least one of SEQ ID NOS:1-3 and 133-134 by less thanabout 30 amino acids.

Certain non-limiting embodiments of the present disclosure are directedto a method of treating or reducing the occurrence of pulmonary fibrosis(such as, but not limited to, idiopathic pulmonary fibrosis) in asubject. In the method, any of the compositions disclosed or otherwisecontemplated herein may be administered to the subject. For example (butnot by way of limitation), the composition may be selected from: (i) acomposition comprising a Fetal-lethal noncoding developmental regulatoryRNA (FENDRR) IncRNA or a fragment or variant thereof and apharmaceutically-acceptable carrier (as described in detail herein aboveor otherwise contemplated herein); or (ii) a composition comprising avector and a pharmaceutically-acceptable carrier, wherein the vectorcomprises a sequence encoding at least about 100 contiguous nucleotidesof a Fetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNAor a fragment or variant thereof (as described in detail herein above orotherwise contemplated herein). In particular (but not by way oflimitation): in (i), the FENDRR IncRNA is encoded by a sequencecomprising at least about 100 contiguous nucleotides of at least one ofSEQ ID NOS:1-3 and 133-134, or a sequence that differs from at least oneof SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids; and in(ii), the sequence comprises at least about 100 contiguous nucleotidesof at least one of SEQ ID NOS:1-3 and 133-134, or a sequence thatdiffers from at least one of SEQ ID NOS:1-3 and 133-134 by less thanabout 30 amino acids.

In the methods of the present disclosure, the compositions may beadministered in therapeutically effective amounts. An effective amountis a dosage of the composition sufficient to provide a therapeuticallyor medically desirable result or effect in the subject to which thecomposition is administered. The effective amount will vary with theparticular condition being treated, the age and physical condition ofthe subject being treated, the severity of the condition, the durationof the treatment, the nature of the concurrent or combination therapy(if any), the specific route of administration, and like factors withinthe knowledge and expertise of the health practitioner. For example, inconnection with methods directed towards treating subjects having acondition characterized by pulmonary fibrosis, an effective amount wouldbe an amount sufficient to mitigate, reduce, modulate, inhibit, orotherwise effectively treat the condition in the subject.

Generally, a therapeutically effective amount will vary with thesubject's age, condition, and sex, as well as the nature and extent ofthe disease in the subject, all of which can be determined by one ofordinary skill in the art. The dosage may be adjusted by the individualphysician or veterinarian, particularly in the event of anycomplication. A therapeutically effective amount is typically, but notlimited to, an amount in a range from 0.1 μg/kg to about 2000 mg/kg, orfrom 1.0 μg/kg to about 1000 mg/kg, or from about 0.1 mg/kg to about 500mg/kg, or from about 1.0 mg/kg to about 100 mg/kg, in one or more doseadministrations daily, for one or more days. If desired, the effectivedaily dose of the active compound may be administered as two, three,four, five, six, or more sub-doses, for example, administered separatelyat appropriate intervals throughout the day, optionally, in unit dosageforms. In some embodiments, the inhibitors are administered for morethan 7 days, more than 10 days, more than 14 days, or more than 20 days.In still other embodiments, the inhibitor is administered over a periodof weeks or months. In still other embodiments, the inhibitor isdelivered on alternate days. For example, the agent may be deliveredevery two days, or every three days, or every four days, or every fivedays, or every six days, or every week, or every month.

The compounds of the presently disclosed inventive concepts may beadministered alone or in combination with one or more additionaltherapies and may be administered by a variety of administration routes.The particular mode selected will depend, of course, upon the compoundselected, the condition being treated, the severity of the condition,whether the treatment is therapeutic or prophylactic, and the dosagerequired for efficacy. The methods of the present disclosure, generallyspeaking, may be practiced using any mode of administration that ismedically acceptable, meaning any mode that produces effective levels ofthe active compounds without causing clinically unacceptable adverseeffects. Non-limiting examples of administration routes that may beutilized in accordance with the present disclosure include oral,topical, transdermal, parenteral, subcutaneous, intranasal,intratracheal, intrabronchial, mucosal, intramuscular, intraperitoneal,intravitreal, and/or intravenous routes, and the like.

For example (but not by way of limitation), the composition can beadministered to the pulmonary tract by any methods known in the art orotherwise contemplated herein. For example, but not by way oflimitation, commercially available devices are known for many differentmethods and mechanisms of delivering various liquid or aerosolizedpharmaceutical formulations to pulmonary tissue, including (but notlimited to), intranasal instillation devices, intratracheal instillationdevices, intratracheal injection devices, dry powder inhalers (DPIs),pressurized metered dose inhalers (pMDIs), nebulizers (such as, but notlimited to, pneumatic (jet) nebulizers and electromechanicalnebulizers), electrohydrodynamic aerosol devices, insufflators,respirators, and the like. These devices can include a single dose ormultiple doses of the compositions of the present disclosure.

In addition, the formulations of the compositions of the presentdisclosure may include one or more additional components/elements thataid in the administration of the compositions, as based upon thedelivery device. For example (but not by way of limitation), pMDIs,DPIs, and nebulizers typically employ one or more propellants to propelthe liquid or cloud of dry powder formulation out of the device, to forman aerosol, and/or to atomize the liquid formulation. Any suitablepropellants/pressurized gas supplies may be utilized. The propellant maytake a variety of forms. For example, the propellant may be a compressedgas or a liquefied gas. Aerosol formulations for use in the subjectmethod typically include (for example, but not by way of limitation)propellants, surfactants, and/or co-solvents. Suitable liquidcompositions comprise the active ingredient in an aqueous,pharmaceutically acceptable inhalant solvent such as (but not limitedto) isotonic saline or bacteriostatic water. Suitable liquidformulations for nasal sprays or nasal drops typically include (forexample, but not by way of limitation) aqueous or oily solutions of theactive ingredient.

When the compositions are formulated for being inhaled, the inhaledformulation may be designed for application to the upper (such as, butnot limited to, the nasal cavity, pharynx, and larynx) and/or lowerrespiratory tract (such as, but not limited to, the trachea, bronchi,and lungs). Different devices and excipients can be used depending onwhether the application is to the upper and/or lower respiratory tractand can be determined by those skilled in the art.

Example

An Example is provided hereinbelow. However, the present disclosure isto be understood to not be limited in its application to the specificexperimentation, results, and laboratory procedures disclosed herein.Rather, the Example is simply provided as one of various embodiments andis meant to be exemplary, not exhaustive.

In the present Example, FENDRR was identified as a down-regulated IncRNAin the lungs of IPF patients. In addition, the functional roles andunderlying mechanisms of FENDRR in pulmonary fibrosis were furtherdetermined. The results of this Example showed that FENDRR inhibited theactivation of lung fibroblasts by binding iron-responsiveelement-binding protein 1 (IRP1) to control iron levels and by competingwith the pro-fibrotic miR-214. This Example also demonstrated thatFENDRR functions as an anti-fibrotic IncRNA in vivo.

Materials and Methods

RNA-seq analyses: Two next-generation RNA sequencing datasets from thelung tissues of IPF patients are publicly available from NCBI's SequenceRead Archive (SRA accession number SRA048904) and NCBI's Gene ExpressionOmnibus (GEO Series accession number GSE52463). These datasets werere-analyzed to identify altered IncRNAs in IPF lungs. RNAs from 3 normaland 3 IPF patient lungs were sequenced in the SRA048904 datasets. RNAsfrom 7 normal and 8 IPF patient lungs were sequenced in the GSE52463datasets. These datasets were re-analyzed to identify altered IncRNAs inIPF lungs. Paired-end reads were directionally mapped to the genomicloci of IncRNA (GRCh37/hg19) by TopHat2(https://ccb.jhu.edu/software/tophat/manual.shtml). CuffDiff analysiswas then performed to identify the dysregulated IncRNAs(cole-trapnell-lab.github.io/cufflinks/). IncRNAs with a log 2-foldchange ≥1 and a False Discovery Rate (FDR)<0.05 were considered to bedifferentially expressed.

IPF lung tissues and RNA isolation: Twenty-seven IPF patient lung tissuesamples were obtained from Lung Tissue Research Consortium (LTRC). Allof the samples were submerged in RNAlater solution and stored at −80° C.before use. Total RNA was isolated from these human lung tissues, murinelung tissues or cells using Tri Reagents (Molecular Research Center,Cincinnati, Ohio). Cytoplasmic and nuclear RNAs were isolated from thecytoplasmic and nuclear fractions of human lung fibroblasts, which wereseparated using a Cytoplasmic & Nuclear RNA Purification Kit (NorgenBiotek Corp, Thorold, ON, Canada) (Catalog #21000). The RNAconcentration and quality were determined with a NanoDrop ND-1000Spectrophotometer (NanoDrop Tech., Rockland, Del.) with an A₂₆₀/A₂₈₀ratio >1.8 and an A₂₆₀/A₂₃₀ ratio >1.7.

Cell culture: HEK 293T cells and human lung fibroblasts (HFL1, CCD-8Lu,and LL29) were purchased from the American Type Culture Collection(ATCC) (Manassas, Va., USA). HEK 293A cells were purchased fromInvitrogen. HFL1 cells were human diploid fibroblasts derived from fetallungs. CCD-8Lu and LL29 cells were human fibroblasts isolated from thelungs of a healthy adult and an IPF patient, respectively. The cellswere grown and maintained with the following media supplemented with 10%fetal bovine serum (FBS): HFL1, and LL29 cells, F12K Medium (Kaighn'sModification of Ham's F-12 Medium); and CCD-8Lu, Eagle's MinimumEssential Medium. Primary human pulmonary fibroblasts (HPF) werepurchased from PromoCell (Heidelberg, Germany). HPF cells were culturedin fibroblast medium with its supplements (PromoCell, Heidelberg,Germany).

Isolation of mouse lung fibroblasts: Primary lung fibroblasts wereisolated from the lungs of saline- or bleomycin-treated mice accordingto a previously described protocol (1). The cells were cultured in DMEMcontaining 10% FBS and used at passages 3-9. Alveolar epithelial type IIcells were also isolated from these mice and differentiated intoalveolar epithelial type I cells, as previously described (2).

Real-Time PCR

Relative real-time PCR: The mRNA and IncRNA expression levels weredetermined by SYBR Green I-based real-time PCR. One microgram ofDNase-treated total RNA was reverse-transcribed into cDNA with randomprimers and oligo dT. The real-time PCR thermal conditions were 95° C.for 10 min, followed by 40 cycles of 95° C. for 15 sec and 60° C. for 60sec. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin wereused as internal controls. The microRNA expression levels weredetermined by Real-time PCR as previously described (3). The sequencesof the primers used for real-time PCR are shown in Table 1.

TABLE 1 Primers for Real-Time PCR SEQ ID Name Sequence NO: HumanNR_033925-FENDDR- CGCACAGACCCAGGATTACTTC 4 FW (variant 1)NR_033925-FENDDR- GCTCCTTATGCAAGCATTCTTC 5 RE (variant 1) ANR_036444-FENDDR- CCCTGCTCCTCTCGAATTTCT 6 FW (variant 2)NR_036444-FENDDR- CCATGCACCAAATCCTTAAAAT 7 RE (variant 2) GTMK522493-FENDDR- CAGAAGCCCCCTCCTGTTATC 8 FW (variant 3) MK522493-FENDDR-AAGAAGCCAAGCCCATTCTGT 9 RE (variant 3) FENDRR-FW (variantGCGCACAGACCCAGGATTT 10 2, 3) FENDRR-RE (variant ACACGGGCAGAGCTGGTTT 112, 3) hCOL1A1-FW CGAAGACATCCCACCAATCAC 12 hCOL1A1-RECAGATCACGTCATCGCACAAC 13 hCOL3A1-FW TGGCTACTTCTCGCTCTGCTT 14 hCOL3A1-RETTCCAGACATCTCTATCCGCAT 15 AG hαSMA-FW GTGTTGCCCCTGAAGAGCAT 16 hαSMA-RECGCCTGGATAGCCACATACAT 17 hGAPDH-FW GAAGGTGAAGGTCGGAGTCAAC 18 hGAPDH-RECATGGGTGGAATCATATTGGAA 19 hACTB-FW GGCACCACACCTTCTACAATGA 20 hACTB-REACAGCCTGGATAGCAACGTACA 21 hU2snRNA-FW CATCGCTTCTCGGCCTTTTG 22hU2snRNA-RE TGGAGGTACTGCAATACCAGG 23 hTFRC-FW ATCCGGTTACTGGGCAATTTC 24hTFRC-RE TCTGTGTCCTCGCAAAAACAG 25 mature miR-214 ACAGCAGGCACAGACAGGCA 26pri-miR-214-FW CCCTTTCCCCTTACTCTCCAA 27 pri-miR-214-REGGATGTTCTGCACAGCAAGT 28 Mouse NR_130109-Fendrr- GAACTCAGGACCTCTGGAAGA 29FW (variant 1) NR_130109-Fendrr- GGTCTGCCTTGTCGTTTTCTT 30 RE (variant 1)NR_045471-Fendrr- TGCTGAATGGAGGCATCTACA 31 FW (variant 2)NR_045471-Fendrr- GCTTGAACCGTCTCTCCTTTG 32 RE (variant 2)FENDRR-FW (variant CACGATCCCAGGTGGACTTG 33 1, 2) FENDRR-RE (variantTGCAGGAGTGAAGGGTGTCTCT 34 1, 2) mCOL1A1-FW ACGCATGGCCAAGAAGACAT 35mCOL1A1-RE TTGTGGCAGATACAGATCAAGC 36 A mCOL3A1-FW CACCCTTCTTCATCCCACTCTT37 mCOL3A1-RE TGACATGGTTCTGGCTTCCA 38 mGAPDH-FW CTCGTCCCGTAGACAAAATGGT39 mGAPDH-RE TGATGGCAACAATCTCCACTTT 40

Absolute real-time PCR: The expression levels of FENDRR transcriptvariants were also determined by using the SYBR Green I-based real-timePCR. Conventional PCR products were amplified and purified with gelextraction, and copy numbers were calculated to construct standardcurves (10³-10⁸ copies). A standard curve was plotted with thresholdcycle (CT) versus the logarithmic value of the gene copy number. The RNAcopy numbers of unknown samples were generated directly from thestandard curves with Sequence Detector software provided by the 7500Fast Real-Time PCR system. All copy numbers were normalized to GAPDHmRNA.

Droplet digital PCR: Droplet digital PCR: The expression of FENDRRvariants were also determined by QX200 AutoDG droplet digital PCR(ddPCR) system (Bio-Rad, Hercules, Calif.) according to themanufacturer's instructions. The ddPCR reaction mixture consisted of theQX200™ ddPCR™ EvaGreen Supermix, forward and reverse primer (200 nM),and 5 μl cDNA in a total reaction volume of 20 μl Droplets weregenerated in 8-well cartridges using the automated droplet generator.After completion of the droplet generation, water-in-oil emulsions weretransferred to a 96-well plate, and PCR was performed using a C1000Touch™ thermal cycler. Thermal cycling conditions were 95° C. for 5 min,followed by 40 cycles at 95° C. for 30 secs, 60° C. for 1 min with afinal 5 min at 90° C. After PCR amplification, fluorescence intensitiesof each droplet from the samples were measured using the QX200 dropletreader. Positive droplets containing amplification products weredistinguished from negative droplets and counted by applying afluorescence amplitude threshold in QuantaSoft software Version1.7.4.0917. QuantaSoft software provides copies of a gene per microliter(copies/μl). The number of copies of a gene per template cDNA wascalculated as copies/μl×20 (μl). The copy number of FENDNR variants werenormalized to that of β-actin. The primers used are listed in Table 1.

Construction of plasmids: FENDRR and miR-214 expression vectors: FENDRRwas amplified by PCR using specific primers (Table 2) from human lungtissue cDNA. Mature miR-214 plus 200 bp flanking sequences at each endwas amplified by PCR using specific primers (Table 2) from human genomicDNA. The fragments were inserted into adenoviral and lentiviral vectorsat XhoI and EcoRI sites, as previously described (4, 5). The controlvector was constructed with a random genomic DNA insert with a length ofapproximately 500 bp that did not contain any known IncRNAs, microRNAs,or mRNAs.

shRNA vectors: All of the shRNAs were designed by the BLOCK-iT™ RNAiDesigner software from Invitrogen (Grand Island, N.Y.). shRNAs wereinserted into the lentiviral pSIH-H1 vector (System Biosciences,Mountain View, Calif.), which utilizes the H1 promoter to drive shRNAexpression. The shRNA sequences are listed in Table 2. A control vectorcontaining scrambled shRNA was purchased from System Biosciences(Mountain View, Calif.).

FENDRR promoter vector: The 5′-flanking region of FENDRR (−1653 to +90)was amplified by PCR using specific primers (Table 2) from human genomicDNA. The fragments were inserted into the luciferase reporter vectorpGL3-Basic (Catalog #E1751, Promega, Madison, Wis.).

miR-214 sensor vector: DNA containing four copies of the miR-214 bindingsite was inserted downstream of the firefly luciferase gene using thepmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega,Madison, Wis.) at the Nhe I and Sal I sites. pmirGLO also contains therenilla luciferase gene for normalization. The control vector wasconstructed with a similar-sized random DNA insert that did not containany known miRNA binding sites. The sensor sequences are listed in Table2.

All of the inserts in the plasmid constructs described above wereconfirmed by DNA sequencing.

TABLE 2 Primers for the Construction of Plasmids SEQ ID Human NO:microRNA sensor CON-sensor- TCGAGGGGTTCACCGATCCTCCACTGCAGTTGG 41 FWTTCCGCCAGCAGACGAGAACTATTTCCTTAAGT TGTGAAGATCTCTTCGGTAGGCCAGCTGGGTTTTAACATG CON-sensor- AATTCATGTTAAAACCCAGCTGGCCTACCGAAG 42 REAGATCTTCACAACTTAAGGAAATAGTTCTCGTC TGCTGGCGGAACCAACTGCAGTGGAGGATCGGTGAACCCC miR-214- TCGAGTCTAACTGCCTGTTCCTGCCTGCTGTTA 43 sensor-FWTTACTGCCTGTGTGAGCCTGCTGTACATACTGC CTGTCCAGGCCTGCTGTACATACTGCCTGTATTGGCCTGCTGTG miR-214- AATTCACAGCAGGCCAATACAGGCAGTATGTAC 44 sensor-REAGCAGGCCTGGACAGGCAGTATGTACAGCAGGC TCACACAGGCAGTAATAACAGCAGGCAGGAACAGGCAGTTAGAC FENDRR overexpression FENDRR-FWTTTCTCGAGCAGACAGCGCGGGCTGGGAG 45 FENDRR-RETTTGGTCTCGAATTGTCCATCGAGTTGTCATGC 46 TT miR-214 overexpressionmiR-214-FW TATCTCGAGTTCTGTTACGCAAATTATCCATG 47 miR-214-RETCTGAATTCATAGGCACCACTCACTTTACTT 48 FENDRR promoter FENDRR-FWTGTTGCTAGCGGGAGGAGGAGGAGGAGGAGGAG 49 FENDRR-RETGTTCTCGAGGGCAGGTCTGCGTGCGAGCC 50 shRNA Smad2-shRNA-GATCCGCCTGATCTTCACAGTCATCATTCAAGA 51 FW GATGATGACTGTGAAGATCAGGCTTTTTGSmad2-shRNA- AATTCAAAAAGCCTGATCTTCACAGTCATCATC 52 RETCTTGAATGATGACTGTGAAGATCAGGCG Smad3-shRNA-GATCCGCAACCTGAAGATCTTCAACATTCAAGA 53 FW GATGTTGAAGATCTTCAGGTTGCTTTTTGSmad3-shRNA- AATTCAAAAAGCAACCTGAAGATCTTCAACATC 54 RETCTTGAATGTTGAAGATCTTCAGGTTGCG FENDRR- GATCCGATTTGCCAGCAACTGCATCATTCAAGA55 shRNA-FW GATGATGCAGTTGCTGGCAAATCTTTTTG FENDRR-AATTCAAAAAGATTTGCCAGCAACTGCATCATC 56 shRNA-RETCTTGAATGATGCAGTTGCTGGCAAATCG IRP1-shRNA-GATCCGCCATTGGATCCTGTACAACCTTCAAGA FW1 GAGGTTGTACAGGATCCAATGGCTTTTTG 57IRP1-shRNA- AATTCAAAAAGCCATTGGATCCTGTACAACCTC 58 RE1TCTTGAAGGTTGTACAGGATCCAATGGCG IRP1-shRNA-GATCCGCAAATTTGTCGAGTTCTTCGTTCAAGA 59 FW2 GACGAAGAACTCGACAAATTTGCTTTTTGIRP1-shRNA- AATTCAAAAAGCAAATTTGTCGAGTTCTTCGTC 60 RE2TCTTGAACGAAGAACTCGACAAATTTGCG IRP1-shRNA-GATCCGCCATTACTAGCTGCACAAACTTCAAGA 61 FW3 GAGTTTGTGCAGCTAGTAATGGCTTTTTGIRP1-shRNA- AATTCAAAAAGCCATTACTAGCTGCACAAACTC 62 RE3TCTTGAAGTTTGTGCAGCTAGTAATGGCG

Production of lentiviruses and adenovirus: Lentiviruses were producedusing the Lenti-X™ HTX Packaging vectors (Clontech, Mountain View,Calif.) in HEK 293T cells. For the production of adenovirus, a pENTRvector was switched into the adenoviral vector pAd/PL-DEST using thegateway technique (Invitrogen). The obtained adenoviral vector waslinearized by Pac I and transfected into HEK 293A cells to producevirus. Adenovirus was further amplified by re-infecting HEK 293A cells.The adenoviruses were concentrated and purified with the AdenovirusStandard Purification ViralKit™ (VIRAPUR, San Diego). The viral titerswere determined by infecting HEK 293T or HEK 293A cells with a series ofdilutions of the viral stock and counting the numbers of virus-infectedgreen fluorescent protein (GFP)-positive cells.

Generation of fibroblasts stably expressing FENDRR: Stable cell linesexpressing FENDRR were generated using a lentiviral FENDRR vectorcoupled with puromycin selection. LL29 cells were treated withlentiviral FENDRR or the virus control at a multiplicity of infection(MOI) of 50. After 48 hours, cells were selected with puromycin at 0.5μg/ml for 1 week until they reached confluence, and the medium waschanged every two days. The stable cell lines were then cultured with amaintenance concentration of puromycin (0.1 μg/ml) and directly used forfurther experiment. The stable cell lines were used at passages 4-5.

Dual luciferase assay: For the promoter luciferase reporter assay, HPFsand HFL1 cells were seeded onto a 96-well plate at a density of 2×10⁴cells per well and transfected with 50 ng of the FENDRR promoterreporter vector and TK plasmid (15 ng), which expresses Renillaluciferase for normalization, using Lipofectamine™ 2000 (Invitrogen). 24hours post transfection, cells were treated with TGβ31 (5 ng/ml). Forthe miR-214 sensor luciferase assay, 1) HEK293T cells were seeded onto a96-well plate at a density of 2×10⁴ cells per well and transfected with5 ng miR-214 sensor and 20, 40, or 60 ng miR-214 expression or controlvector and 150 ng FENDRR expression or control vector usingLipofectamine™ 2000. 2) LL29 cells stably expressing FENDRR or controlwere seeded onto a 96-well plate at a density of 2×10⁴ cells per welland transfected with 50 ng miR-214 sensor or control sensor usingLipofectamine™ 2000. The cells were collected 48 hours aftertransfection, and firefly and Renilla luciferase activities weremeasured using the Dual-Luciferase® Reporter Assay System (Promega,Madison, Wis.). The results are presented as the ratio of firefly toRenilla luciferase activities.

RNA pulldown and mass spectrometry analysis: FENDRR with the SP6promoter were PCR-amplified from FENDRR overexpression vector to serveas DNA template. Antisense FENDRR was amplified as a control (Table 3).The DNA templates were in vitro transcribed to obtain FENDRR and controlRNA by using an in vitro transcription kit (ThermoFisher ScientificCatalog #AM1330). The RNAs were labeled using a Pierce RNA 3′ EndDesthiobiotinylation Kit, and the proteins that interacted with theFENDRR RNA were immunoprecipitated using a Pierce Magnetic RNA-ProteinPull-Down Kit (Thermo Scientific Catalog #20163 and 20164). The RNApull-down protein samples were analyzed by mass spectrometry (LTQOrbitrap XL).

TABLE 3 Primer Sequences for RNA Pulldown SEQ ID Name Sequence NO:SP6-FENDRR-FW ATTTAGGTGACACTATAGAAGAGCA 63 (variant 3) GACAGCGCGGGCTGSP6-FENDRR-RE ATATCTATATATGCAAATTAGATGT 64 (variant 3) CTAAATCTATATTCGSP6-anti-sense ATTTAGGTGACACTATAGAAGAGCA 65 FENDRR-FWAATTAGATGTCTAAATCTATATTC (variant 3) SP6-anti-sense CAGACAGCGCGGGCTGGGAG66 FENDRR-RE (variant 3)

RNA immunoprecipitation (RIP) assay: RIP was carried out as describedpreviously (6). Confluent LL29 cells in 10-cm dishes were harvested bytrypsin digestion and were lysed in RIP buffer. The cell lysate wascentrifuged at 10,000×g for 15 minutes, and the supernatant wascollected. Ten μg of rabbit anti-aconitase 1 (ACO1) (also named IRP1)antibodies (catalog no. Ab126595, Abcam, Boston, Mass.) or IgG controlantibodies were added to 50 μl supernatant, and the mixture wasincubated overnight with rotation at 4° C. Forty microliters of proteinA/G beads were added and incubated at 4° C. for 1 hour with gentlerotation. The beads were washed three times with ice-cold RIP buffer andthree times with PBS. The co-precipitated RNAs were isolated from there-suspended beads in 1 mL Tri Reagents (Molecular Research Center). Theamount of FENDRR in the co-precipitated RNAs was determined by real-timePCR and was calculated with the equation 2^(−Ct). Enrichment fold wascalculated over IgG control.

Cross-linking immunoprecipitation and qPCR (CLIP-qPCR) analysis:CLIP-qPCR was performed as previously described (7). LL29 cells werecultured in 10-cm dishes until confluence. Cross-linking was performedby irradiating the cells on dishes with 150 mJ/cm² of UVA inSpectrolinker. The cell pellets were resuspended in three volumes(relative to pellet size) of NP-40 lysis buffer (7). Cell lysates weredigested with 1 U/μl of RNase T1 (ThermoFisher Scientific Catalog#AM2280) for 2, 4, 8, and 12 min. The samples treated with RNAse T1 for2 min having RNAs partially digested in the 100- to 300-nt range in 1.5%formaldehyde agarose gel were selected. One ml of cell lysates was addedto the Sepharose beads coated with 10 μg of normal IgG or anti-IRP1(ACO1) antibodies and incubated for 3 hours at 4° C. The beads werewashed three times with NP-40 lysis buffer and incubated with 20 unitsof RNase-free DNase I in 100 μl NP-40 lysis buffer for 15 min at 37° C.The beads were then incubated with 0.1% SDS and 0.5 mg/ml Proteinase Kfor 15 min at 55° C. RNA was isolated with acidic phenol extraction andethanol precipitation. Real-time PCR was used to quantify the relativeabundance of overlapping segments spanning the FENDRR RNA. For primerdesign, FENDRR was divided into 200-nt overlapping intervals so that theamplified products covered all of the full-length transcript. Thesequences of the primers used for real-time PCR are shown in Table 4.

TABLE 4 Primers for CLIP-qPCR SEQ Primer ID Region name Primer sequenceNO: 134-257 FW1 CGAAAGGTGGTGCCGAGAG 67 134-257 RE1 TCCGTTTGCATCCAACATTGT68 195-294 FW2 GCACAGACCCAGGATTTGTG 69 195-294 RE2 CAGAGCTGGTTTTGACAGTGA70 241-382 FW3 TGTTGGATGCAAACGGATTTG 71 241-382 RE3CCCTCTCTGGTCTTCAGTTTCT 72 301-413 FW4 TCTTCCGAAGATACCAAGTGAAA 73 301-413RE4 GTCAGTTGTGCCAAACTGAGT 74 373-511 FW5 CCAGAGAGGGTGAGTGGTTTA 75373-511 RES TGCAGTTCCTGTAGGTCAGAA 76 474-587 FW6 CCCTGCTCCTCTCGAATTTCT77 474-587 RE6 TTTCTGGTTATCTACGACTGCAT 78 540-704 FW7CCACTGCATTTTGGCATGATT 79 540-704 RE7 GCACACTGCTCAGAGAATGTG 80 649-768FW8 CCACCAATTGGCTCGATGAG 81 649-768 RE8 GTGACCTGTGAGTGGCGATAA 82 732-857FW9 CAGAAGCCCCCTCCTGTTATC 83 732-857 RE9 AAAGAAGCCAAGCCCATTCTG 84837-962 FW10 CAGAATGGGCTTGGCTTCTTT 85 837-962 RE10 AGCCTATGTCCCATCAACAGT86  909-1058 FW11 CGTTTGTTCATTTTCACCACCAT 87  909-1058 RE11CCTCCAACAGAAATGCATGCA 88 1001-1149 FW12 CCAGCTGGAGACTGGTATATGT 891001-1149 RE12 GCACCAAATCCTGAGAAAGAAGA 90 1086-1227 FW13CAGACCACCCTCAAATTGAGT 91 1086-1227 RE13 CCAGTTGAGCCTCTGAATGAC 921204-1345 FW14 GCAGTCATTCAGAGGCTCAAC 93 1204-1345 RE14CACAGGGCAAGGATACAGAGA 94 1312-1445 FW15 TCCTCAGCTCACCTCTCTGTA 951312-1445 RE15 AGTGACAGTCTGGGACATCTG 96 1419-1549 FW16AGGATTCAGATGTCCCAGACT 97 1419-1549 RE16 AAGATGATCCCCAACAATGCT 981496-1642 FW17 CAGGAATGGGTGGCATATGC 99 1496-1642 RE17TCTGAGCACACAATCCACTCT 100 1596-1738 FW18 AGGAAGTCCACTATGCTTGCT 1011596-1738 RE18 CCTTGATTCACAATGGCTCAGT 102 1715-1847 FW19GCACTGAGCCATTGTGAATCA 103 1715-1847 RE19 CTGGTTTGTGGAGGAGAGAGT 1041812-1933 FW20 TTGGATTCCAGGGGCACTCT 105 1812-1933 RE20GCCACTCACATTGTTGCTGAA 106 1903-2043 FW21 GATCCTGCTGTTCAGCAACAA 1071903-2043 RE21 ACTGCAAACCAAAAGGTGCTT 108 1995-2118 FW22CCGGTCACTTCACGATGACA 109 1995-2118 RE22 AACTTCTCCAAGCACAAAGTGT 1102071-2246 FW23 CGGCTTCAGGAATTCACAGAA 111 2071-2246 RE23CCTAGGTTGTCTCCACTGCTA 112 2178-2319 FW24 GGAGGTCCTGTGTATGAGGAT 1132178-2319 RE24 ACTGCTGAACACACTTTTCCT 114 2272-2437 FW25GAGCCCTACAGCAGTGAAAAG 115 2272-2437 RE25 ACCTGGGCATTTACCTTCAGA 1162372-2532 FW26 GAACTAGGGTAGAGGCACTAGAG 117 2372-2532 RE26CCCCCTTTCCTGACTACTTAACT 118 2481-2649 FW27 TCACCTGACCTCTGTGTTTACA 1192481-2649 RE27 GCATATTCATGGCAGCTGGTA 120 2580-2739 FW28AGTCCCCAAAAACAGGTGGAA 121 2580-2739 RE28 ACAGGCATGTTTTCTTTCCTAGA 1222654-2816 FW29 GCTGCTGGCTAGTGATAAATAAC 123 2654-2816 RE29CTCTGTGCCCATCTCTACCAT 124 2758-2901 FW30 AGCTTAGACAAAGATGGGCAAA 1252758-2901 RE30 CATTCCCCGGCTTGTAAAGG 126 2835-2991 FW31CACAAACAGAAGGCCAAGTGA 127 2835-2991 RE31 AAAGGTGATGAAGGGCCAGTT 1282949-3085 FW32 CTCCGTCAGAGTCTCCAGAAG 129 2949-3085 RE32CGTTTTCTGTGGCAACCATAAC 130 3007-3174 FW33 CAGAACTGGGCAAGAAAATGTTT 1313007-3174 RE33 GTTGTCCATCGAGTTGTCATG 132 *The primers detect humanFENDRR variant 3.

Iron levels and aconitase activity assay: Iron levels in the fibroblastsand lung tissues were determined by using an Iron Assay Kit purchasedfrom Sigma (Catalog #MAK025) and expressed as nmol/mg protein. Aconitaseactivity was measured by using an Aconitase Enzyme Activity MicroplateAssay Kit purchased from Abcam (Cata #ab109712) according to themanufacturer's instructions and expressed as nmol/min/mg protein.

Gel contraction activity: Gel contraction activities were measured aspreviously described (8). LL29 cells stably expressing FENDRR or controlvector were split into 6-well plates (200,000 cells/well). The cellswere treated with TGFβ1 (5 ng/ml) for 72 hours. Then, the cells weremixed with collagen I (MilliporeSigma, Burlington, Mass.) to a finaldensity of 1×10⁵ cells per ml and a final concentration of 1 mg/ml ofcollagen I with or without 5 ng/ml of TGFβ1. Gel images were taken at 24hours and quantitatively analyzed using Image J software.

Western blotting: Protein samples were separated on 12%SDS-polyacrylamide gels and transferred onto nitrocellulose membranes.Primary antibodies and dilutions used for Western blotting included:rabbit anti-SMAD2 (catalog no. 3122S, 1:1000 dilution, Cell signaling,Beverly, Mass.); rabbit anti-SMAD3 (catalog no. 9523S, 1:1000 dilution,Cell signaling); rabbit anti-COL1A1 (catalog no. sc-8784-R, lot #J3013,1:500 dilution, Santa Cruz Biotechnology, California, CA); rabbitanti-COL3A1 (catalog no. sc-8780-R, lot #L1010, 1:500 dilution, SantaCruz Biotechnology); mouse anti-α-SMA (catalog no. A2547, 1:1000dilution, Sigma, St Louis, Mo.); and mouse anti-GAPDH (catalog no.ab181602, 1:4000 dilution, Abcam, Boston, Mass.). The secondaryantibodies (horseradish peroxidase-conjugated Immuno-Pure anti-rabbit ormouse IgG; HOUR+L) were used at a dilution of 1:5000. The blots weredeveloped with Super Signal West Pico Luminol Enhancer solution andSuper Signal West Pico Stable Peroxidase solution (ThermoFisherScientific).

Immunocytochemistry: LL29 cells stably expressing FENDRR or controlvector were cultured and maintained with 0.1 μg/ml of puromycin. Cellswere split into 8-chamber slides at a density of 4,000 cells per chamberfor 24 hours. The cells were stimulated with 5 ng/ml of TGF-β1 for 48hours, and then the cells were fixed for immunocytochemical analysiswith an anti-α-SMA antibody (1:500) (Sigma) and Alexa 546-conjugatedsecond antibody (1:300) (Molecular Probes, Eugene, Oreg.). Nuclei werestained with Hoechst 33342 (2 μg/ml).

A mouse model of bleomycin-induced pulmonary fibrosis: The animalprocedures were approved by the Institutional Animal Care and UseCommittee at Oklahoma State University. C57BL/6 male mice (8-10 weeksold) were randomly divided into four groups: saline (Sal) and controladenovirus (AdCON), saline and FENDRR adenovirus (AdFENDRR), bleomycin(Bleo) and control adenovirus, and bleomycin and FENDRR adenovirus. Onday 0, 60 μl of FENDRR or control adenovirus (5×10⁹ infectious units(IU) per mouse) were delivered into the lung intranasally. On day 1, 60μl of bleomycin (1 U per kg body weight) (Sigma cat #B8416-15UN) orsaline (Sal) was delivered intranasally. On day 14, respiratorymechanics were determined using the FlexiVent (Scireq, Montreal,Canada). Then, the left lung was collected for RNA, protein and collagencontent analyses. The right lung was fixed in 4% paraformaldehyde forhistological analysis. The degree of fibrosis in the mouse lung wasquantitated using an Ashcroft score in a blinded manner following thepublished method (9).

Hydroxyproline Assay: The amount of collagen in the lung tissues wasdetermined by the hydroxyproline assay according to the manufacturer'sprotocol (QuickZyme Biosciences, Netherland) and expressed as μg per mglung tissue.

Statistical analysis: The data presented in the figures represent themeans±standard error (SEM). Statistical analyses were performed usingStudent's t-test for two-group comparisons, and Analysis of variance(ANOVA), followed by Tukey's HSD test or Fisher's LSD test, for multiplecomparisons. A p-value <0.05 was considered to be significant.

Results

FENDRR is down-regulated in fibrotic lungs and fibroblasts.

There are 2 publicly available RNA_seq datasets from IPF patient lungs:SRA048904, 3 IPF cases and 3 controls (34), and GSE52463, 8 IPFs and 7controls (35). To identify dysregulated IncRNAs in the lungs of IPFpatients, the datasets were re-analyzed for IncRNA expression. 174 (80up and 94 down) and 56 (6 up and 50 down) dysregulated IncRNAs in IPFlungs were identified from the SRA048904 and GSE52463 datasets,respectively. Of these, 7 down-regulated and zero up-regulated IncRNAswere shared between the 2 datasets (FIG. 1, panel A; Table 5). FENDRRwas selected for further studies for the following reasons: (i) FENDRR,but not other IncRNAs (except LINC00961) is conserved between humans andmice; (ii) FENDRR had the fifth and third highest expression among allof the IncRNAs in normal human lungs and human pulmonary fibroblasts(HFPs) (data not shown); and (iii) FENDRR is known to be essential forlung development (21, 23).

TABLE 5 Seven Common Differentially Expressed IncRNAs in IPF Lungs inthe 2 Datasets SRA048904 dataset GSE52463 dataset Gene name Control IPFP value Gene name Control IPF P value RP11-253E3.3 28.11 13.37 0.00165RP11-253E3.3 11.07 4.08 0.0007 RP11-38P22.2 17.53 8.01 0.00080RP11-38P22.2 30.14 6.44 0.00005 LINC00961 38.11 15.44 0.00025 LINC0096111.74 3.06 0.0006 AC093110.3 107.91 40.01 0.00005 AC093110.3 121.7941.46 0.00025 FENDRR 344.53 89.47 0.00845 FENDRR 147.76 29.19 0.00005RP1-249H1.4 8.86 1.94 0.00005 RP1-249H1.4 5.70 1.55 0.00005 LINC0055118.55 2.30 0.00005 LINC00551 7.88 1.00 0.0005

The down-regulation of FENDRR in the lungs was confirmed using real-timePCR in an independent cohort of 27 patients with IPF from the LungTissue Research Consortium (LTRC) and 6 human adult normal lung tissuesfrom BioChain (0.017±0.003 in normal vs. 0.0045±0.001 in IPF) (FIG. 1,panel B). Consistent with these findings using human lung tissues, theFendrr level in mouse lung tissues with bleomycin-induced pulmonaryfibrosis was 40.3±2.4% of the control mouse lungs (FIG. 1, panel C).Fendrr down-regulation was also observed in the fibroblasts, but not inthe AEC I and AEC II isolated from the bleomycin-treated mice (FIG. 1,panel D).

Human FENDRR has two annotated splicing variants, transcript variant 1(NR_036444; SEQ ID NO:1) and transcript variant 2 (NR_033925; SEQ IDNO:2) in the UCSC Genome Browser (http://genome.ucsc.edu). When it wasattempted to clone transcript variant 2 using human lung tissue cDNA, itwas found that the cloned FENDRR was 531 bp longer than the transcriptvariant 2, which is 2,693 bp long (FIG. 2). This was named FENDRRtranscript variant 3. The cDNA sequence of variant 3 was submitted toGenBank and assigned the accession number, MK522493.1 (SEQ ID NO:3).Using absolute real-time PCR, it was found that the FENDRR transcriptvariant 3 was the major transcript in LL29 and HPF fibroblasts (FIG. 1,panel E). Using droplet digit PCR, which provides highly sensitive andabsolute quantitation of gene expression, it was found that the FENDRRtranscript variant 3 was also the major transcript in normal and IPFhuman lungs and was reduced in IPF lungs compared to normal lungs (FIG.1, panel F). Furthermore, variants 1 and 2 were also essentiallyundetectable in normal and IPF lungs even using the highly sensitivedroplet digital PCR technique (FIG. 1, panel F).

There are two mouse Fendrr variants, which are highly homologous (FIG.2). The variant 1 was expressed much higher than variant 2 in the mouselungs. Both transcripts were decreased by bleomycin treatment (FIG. 1,panel G). Human FENDRR transcript variant 3 is positionally conserved tomouse Fendrr transcript variant 1 (FIG. 2) and were used for in vivomouse studies.

Unless noted, a primer pair common to human transcript variants 2 and 3(FIG. 2 and Table 1) were used for detecting human FENDRR expression inthis Example. This primer pair was designed and used before the humanFENDRR transcript 3 was discovered. Because the human transcript variant2 is essentially undetectable in human lungs and human fibroblasts (FIG.1, panels E and F), this primer pair mainly detects the human transcriptvariant 3.

TGFβ1 inhibits FENDRR expression via Smad3 in pulmonary fibroblasts.

To assess whether TGFβ regulates FENDRR expression, HPFs were treatedwith TGFβ1, and FENDRR levels were determined. TGFβ1 reduced FENDRRexpression by 42% as measured using the common primer pair to thetranscript variants 2 and 3 (FIG. 3, panel A). Similar results wereobserved using the primer pair specific to the variant 3 and dropletdigital PCR (FIG. 3, panel B). Promoter reporter luciferase assaysindicated that TGFβ1 decreased the FENDRR promoter activity in primaryHPFs and HFL1 fibroblasts (FIG. 3, panel C). Next, it was determinedwhether the TGFβ1-mediated inhibition of FENDRR expression occurredthrough Smad transcription factors using RNA interference. Thelentiviral shRNA treatment resulted in 79% and 98% decreases in theprotein expression levels of Smad2 and Smad3 in LL29 fibroblasts,respectively (FIG. 3, panel D). The silencing of Smad3 but not Smad2partially reversed the TGFβ1-mediated inhibition of FENDRR expression(FIG. 3, panel E). These results indicate that TGFβ1-SMAD3 signalingcontributes to the down-regulation of FENDRR in fibrotic lungs.

FENDRR Inhibits Fibroblast Activation

FENDRR was overexpressed in IPF LL29 fibroblasts using a lentiviralFENDRR (transcript variant 3) vector coupled with puromycin selection togenerate stable cell lines and evaluated the effects of FENDRRoverexpression on fibroblast activation. A 13-fold increase in FENDRRlevels was observed in the FENDRR-overexpressing cells compared to thevirus control (VC) cells (FIG. 4, panel A). FENDRR overexpressionreduced TGFβ1-induced mRNA and protein expression of α-SMA, COL1A1, andCOL3A1 (FIG. 4, panels B and C). Furthermore, FENDRR overexpressionreduced TGF-β1-induced contractile activity, as determined by thecollagen gel assay and stress fiber formation (FIG. 4, panels D and E).In contrast, knockdown of FENDRR using shRNA increased the mRNA andprotein levels of α-SMA and the collagens in HPF normal lung fibroblasts(FIG. 4, panels F and G). These results indicated that FENDRR inhibitsTGFβ-induced fibroblast activation.

FENDRR Interacts with Iron-Responsive Element-Binding Protein 1

As a first step in exploring the mechanisms of FENDRR activity, thesubcellular localization of FENDRR was determined by measuring theFENDRR levels in the cytoplasmic and nuclear fractions of primary HPFsand LL29 fibroblasts extracted using a Cytoplasmic & Nuclear RNAPurification Kit. FENDRR in both cells had a low ratio of nuclear tocytoplasmic levels (approximately 0.2), similar to those of cytoplasmicGAPDH and ACTB (β-actin) but in contrast to that of nuclear RNA U2(>2.5) (FIG. 5, panel A), indicating that FENDRR is primarily located inthe cytoplasm of fibroblasts.

As a cytoplasmic IncRNA, the inventors hypothesized that FENDRR mayperform its functions by interacting with cytoplasmic proteins. Toidentify such protein partners, RNA pulldown-coupled mass spectrometryanalysis was performed. 29 proteins were enriched in the FENDRRpull-down group with a fold change greater than 2 and a FDR value lessthan 0.05 compared to the control RNA group (Table 6). These proteinsincluded iron-responsive element-binding protein 1 (IRP1) [also namedaconitase1 (ACO1)], which controls iron homeostasis by binding to theiron-responsive element (IRE) of mRNAs related to iron transport andstorage (36). Since iron overload is associated with fibrosis (37), IRP1was selected for further study. First, the interaction of IRP1 andFENDRR in lung fibroblasts was validated. RNA immunoprecipitationanalysis showed the robust enrichment of FENDRR in an IRP1-interactingRNA fraction compared to that of an IgG control (FIG. 5, panel B).Moreover, CLIP-qPCR analysis was performed to determine the interactionregion between FENDRR and IRP1. The results showed that the primarybinding region between FENDRR and IRP1 was located at the 1,419-1,549 bpregion (FIG. 5, panel C). RNA secondary structures of FENDRR, predictedusing IPknot software, are shown in FIG. 6. The region of FENDRR thatinteracts with IRP1 involves the integration and formation of twoindependent RNA structures.

TABLE 6 FENDRR Binding Proteins Spectrum count of Spectrum count ofProtein Control FENDRR Fold Name Description Means SE Means SE changeENOA Alpha-enolase 0.00 0.00 0.74 0.74 N/A IRP1 iron-responsiveelement-binding protein 1 0.00 0.00 9.60 0.79 N/A (Cytoplasmic aconitatehydratase) GRSF1 Isoform 2 of G-rich sequence factor 1 0.00 0.00 2.100.16 N/A CNBP Isoform 2 of Cellular nucleic acid-binding protein 0.090.09 2.08 0.88 22.51 ACINU Apoptotic chromatin condensation inducer 0.190.09 2.70 1.36 14.44 in the nucleus FXR2 Fragile X mental retardationsyndrome-related 0.37 0.37 2.78 1.31 7.53 protein 2 ZO1 Isoform Short ofTight junction protein ZO-1 0.19 0.09 1.40 0.55 7.51 HNRH3 Isoform 2 ofHeterogeneous nuclear 0.37 0.19 2.73 0.66 7.30 ribonucleoprotein H3SRSF9 Serine/arginine-rich splicing factor 9 0.93 0.49 6.53 0.41 7.00ECI2 Isoform 2 of Enoyl-CoA delta isomerase 2, 0.37 0.37 2.46 1.23 6.68mitochondrial RBM25 RNA-binding protein 25 1.03 0.56 6.65 1.06 6.49PRP31 U4/U6 small nuclear ribonucleoprotein Prp31 1.84 0.29 7.05 1.513.84 CCAR1 Isoform 2 of Cell division cycle and apoptosis 1.59 0.80 5.710.96 3.59 regulator protein 1 SRRM2 Serine/arginine repetitive matrixprotein 2 2.20 0.59 7.10 1.04 3.22 IBP7 Isoform 2 of Insulin-like growthfactor-binding 1.16 0.66 3.69 0.60 3.16 protein 7 TRA2A Transformer-2protein homolog alpha 3.55 1.83 10.11 0.54 2.85 RU17 U1 small nuclearribonucleoprotein 70 kDa 5.98 1.11 16.81 0.03 2.81 RBM14 RNA-bindingprotein 14 4.87 1.75 12.17 2.39 2.50 K6PP 6-phosphofructokinase type C1.97 1.07 4.83 1.87 2.45 RNPS1 RNA-binding protein with serine-richdomain 1 1.28 0.65 3.07 0.39 2.41 DHE3 Glutamate dehydrogenase 1,mitochondrial 4.66 1.35 11.19 0.90 2.40 FHL2 Four and a half LIM domainsprotein 2 3.73 2.23 8.78 3.60 2.35 SRSF4 Serine/arginine-rich splicingfactor 4 3.71 0.82 8.72 0.77 2.35 HNRH1 Heterogeneous nuclearribonucleoprotein H 2.35 0.74 5.50 0.93 2.34 PURB Transcriptionalactivator protein Pur-beta 2.72 0.62 6.20 1.20 2.28 SNR40 U5 smallnuclear ribonucleoprotein 40 kDa protein 1.88 0.64 4.22 1.32 2.25 BUB3Isoform 2 of Mitotic checkpoint protein BUB3 3.43 1.12 7.42 1.81 2.17GRP75 Stress-70 protein, mitochondrial 2.21 1.12 4.72 1.57 2.13 SRPK2Isoform 2 of SRSF protein kinase 2 3.16 0.87 6.52 2.35 2.06

FENDRR Controls Iron Levels by Suppressing IRP1

IRP1 is a dual functional protein, with iron regulatory ability andaconitase activity (36, 38). When cellular iron levels are low, IRP1binds an iron-responsive element (IRE) in either the 3′-untranslatedregion (UTR) or 5′-UTR of an mRNA to regulate transport and storage ofiron. However, when cellular iron levels are high, IRP1 functions as thecytoplasmic isoform of aconitase to catalyze the interconversion ofcitrate into isocitrate through cis-aconitate. FENDRR overexpressionreduced the iron level in LL29 fibroblasts, as determined using an IronAssay Kit (FIG. 7, panel A), and the aconitase activity, as measuredusing an Aconitase Enzyme Activity Microplate Assay (FIG. 7, panel B).IRP1 controls iron homeostasis by binding the IRE of mRNAs related toiron transport and storage, including transferrin receptor 1 (TFRC).FENDRR overexpression reduced TFRC mRNA levels (FIG. 7, panel C). Theiron levels in fibroblasts isolated from bleomycin-treated mice werehigher than those isolated from control mice (FIG. 7, panel D).

To determine whether FENDRR still regulates cellular iron levels whenIRP1 is absent, IRP1 was knocked-down using lentiviral shRNAs, and theeffects of IRP1 knock-down on cellular iron levels in theFENDRR-overexpressing fibroblasts was examined. IRP1 protein expressionwas effectively reduced by a pool of three IRP1 shRNAs (FIG. 7, panelE). Then, cellular iron levels in FENDRR-overexpressed and IRP1-knockeddown fibroblasts was measured in the iron-free medium, where IRP1 bindswith iron metabolism mRNAs, and the iron-supplemented medium, where IRP1exists in a free form in cytoplasm. In the iron-free medium, FENDRRreduced cellular iron levels by 64%, and the knock-down of IRP1 rescuedFENDRR-mediated reduction in cellular iron levels (FIG. 7, panel E).However, in the iron-supplemented medium, FENDRR reduced only 33% ofcellular iron levels, and there were no differences in cellular ironlevels between control shRNA and IRP1 knockdown conditions. Theseresults indicate that FENDRR likely competes with the binding of IRP1 toiron metabolism genes under a low cellular iron level.

Iron is Required for Fibroblast Activation

Iron overload has been shown to be associated with lung, liver, andrenal fibrosis (37, 39). Therefore, the effects of iron on lungfibroblast activation were evaluated. Iron depletion by treatment withthe iron chelator desferrioxamine (DFO) suppressed the TGFβ1-inducedmRNA expression of α-SMA, COL1A1, and COL3A1 in human lung LL29fibroblasts (FIG. 7, panels F-H). These results indicated that iron isrequired for TGFβ-induced fibroblast activation.

FENDRR Competes with Pro-Fibrotic miR-214

One of the mechanisms for IncRNA function involves sponging ofmicroRNAs. To determine whether FENDRR could act as a competingendogenous RNA (ceRNA) or molecular sponge, RNAhybrid was used topredict potential microRNA binding sites; six binding sites formiR-214-3p on FENDRR were found (1028-1055, 1661-1676, 1876-1914,2132-2147, 2698-2743, and 3010-3037) (FIG. 8). To validate thepredication experimentally, a miR-214 sensor consisting of the fireflyluciferase gene and 4 copies of miR-214 binding sites was constructedusing the pmirGlo reporter vector. The miR-214 sensor activity wasinhibited by endogenous miR-214 in the LL29 fibroblasts, and FENDRRoverexpression increased the activity of the miR-214 sensor (FIG. 9,panel A). Then, it was determined whether FENDRR directly competes withmiR-214. Transfection of HEK293T cells with a FENDRR expression vectorincreased the miR-214 sensor activity. However, co-transfection with amiR-214 expression vector reduced the sensor activity in both controland FENDRR overexpression groups in a miR-214-dose-dependent manner(FIG. 9, panel B). These results indicate that FENDRR competes withmiR-214.

miR-214 is up-regulated in the fibrotic lung tissues of IPF patients(40) and in other fibrotic tissues (41-43). miR-214 functions as apro-fibrotic agent in the kidneys, liver, and heart (41-43). miR-214 wasoverexpressed in LL29 fibroblasts with a lentiviral vector, and theactivation of fibroblasts was examined. miR-214 increased TGFβ1-inducedCOL1A1 mRNA and protein expression (FIG. 9, panels C and D).

Next, the relationship between iron levels and miR-214 expression wasdetermined. Iron depletion reduced primary and mature miR-214 expressionin LL29 cells (FIG. 9, panel E), indicating that iron overload increasesmiR-214 expression.

FENDRR Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice

Since FENDRR inhibits lung fibroblast activation in vitro, the effectsof FENDRR overexpression in mouse lungs on bleomycin-induced pulmonaryfibrosis in vivo was examined. Adenovirus-mediated gene transfer wasalso used to overexpress human FENDRR transcript variant 3, which ispositionally conserved to mouse fendrr major transcript variant 1.1.7-fold and 3.5-fold increases in FENDRR expression in the lungs ofsaline control- and bleomycin-treated mice, respectively, were observed(FIG. 10, panel A). Histopathological analysis showed reduced fibrosisin the FENDRR-treated group (FIG. 10, panel B). Quantitation of lungfibrosis in a blinded manner revealed that increased FENDRR expressionsignificantly decreased the Ashcroft score (FIG. 10, panel C).Furthermore, increased FENDRR expression inhibited lung collagen levels,as measured by hydroxyproline assay (FIG. 10, panel D), and reducedbleomycin-induced COL1A1 and COL3A1 mRNA expression (FIG. 10, panels Eand F) and protein expression of COL1A1 (FIG. 10, panels G and H).FENDRR also decreased elastance (Ers), indicating an improvement in lungfunction (FIG. 10, panel I).

DISCUSSION

Currently, idiopathic pulmonary fibrosis remains a serious humandisease. The lack of clarity surrounding the pathogenesis of IPF hasresulted in a lack of effective treatments. In the current study, it wasdiscovered that FENDRR was down-regulated in the fibrotic lungs of IPFpatients and bleomycin-treated mice. FENDRR expression was regulated byTGF-β/Smad3 signaling. Functionally, FENDRR reduced pulmonary fibrosisby inhibiting fibroblast activation through decreasing cellular ironlevel via interactions with IRP1 and acting as a ceRNA for profibroticmiR-214 (FIG. 11).

Fendrr is highly expressed in the adult lung compared with othertissues, and it is confined to the mesenchyme in the developing lungs atE14.5 and E18.5 (44). However, at E9.5, Fendrr is restricted to thecaudal end of the lateral plate mesoderm (22). How FENDRR is regulatedremains unknown. In the present Example, it was found that FENDRR isdown-regulated in fibrotic fibroblasts via TGFβ1-SMAD3 signaling.Down-regulation of FENDRR has also been observed in gastric cancer (24).TGFβ-SMAD2/3 signaling has been shown to contribute to the pathogenesisof pulmonary fibrosis (45-47). Our current studies demonstrated thatTGFβ1 inhibited FENDRR promoter activity. Furthermore, knockdown ofSmad3, but not Smad2, reversed TGβ31-mediated reduction of FENDRRexpression, supporting that TGFβ1-SMAD3 signaling contributes to theFENDRR down-regulation by TGFβ1.

Among non-coding RNAs, microRNAs have been extensively studied in IPF.However, little is known regarding the roles of IncRNAs in IPF. In thisExample, FENDRR was found to have anti-fibrotic functions. In in vitrostudies, it was revealed that FENDRR inhibited TGF-β-induced fibroblastactivation, as demonstrated by the inhibition of collagen synthesis;reduced α-SMA mRNA and protein expression; and decreased contractileactivity and stress fiber formation. It is noted that FENDRRoverexpression had little effects on these parameters under the basalconditions (without TGFβ). The possible explanation is that FENDRR mayregulate the factors involved in the TGFβ signal pathway.

In in vivo studies, this Example further revealed thatadenovirus-mediated FENDRR overexpression reduced collagen content andfibrosis in the lungs in response to bleomycin and improved pulmonaryfunction. Pulmonary fibroblasts express low levels of Coxsackie-virusand adenovirus receptor (CAR). It raises a question whether adenoviruscan deliver a gene to fibroblasts. It has been reported that adenoviralvector can be used to deliver ET1 or Fas to mouse and human lungfibroblasts in vitro, respectively (48, 49). Thus, it is possible thatthis may occur in vivo. However, the possibility that FENDRR is alsodelivered to the respiratory epithelial cells, which in turn affectspulmonary fibroblasts, was not excluded.

The inventors discovered that FENDRR was preferentially localized in thecytoplasm of adult lung fibroblasts. FENDRR has been previously shown topredominantly localize in the nucleus during murine lung development(22). However, cellular localization of IncRNAs can be changed underdifferent physiological conditions. For example, IncRNA GASStranslocates from the cytoplasm into the nucleus with the glucocorticoidreceptor in response to dexamethasone treatment (50). Thus, shifting ofFENDRR between the nucleus and cytoplasm might occur during development.

In a previous study, FENDRR was shown to function in development viaepigenetic control mechanisms (20). FENDRR increased the PRC2 occupancyby forming dsDNA/RNA triplexes at target regulatory elements (20). Here,a novel mechanism of FENDRR activity in inhibiting fibroblast activationwas demonstrated, i.e., regulating cellular iron levels. Iron is a traceelement indispensable for nearly all living organisms, as itparticipates in a variety of biological processes, including electrontransport (51, 52), oxygen transport (53), and DNA synthesis (54).However, excessive iron can result in tissue damage due to the formationof free radicals (55, 56). Abnormal iron homeostasis causes a broadspectrum of human diseases. A number of studies has indicated that ironis associated with pulmonary fibrosis. For example, iron deficiencyreduces the severity of bleomycin-induced pulmonary fibrosis inhamsters, possibly due to reduced iron-catalyzed oxygen radicalformation, and lipid peroxidation (57). An accumulation of iron aftersilica instillation causes fibrotic lung injury in rats (58).Mobilization of iron from asbestos with a high percentage of iron canenhance collagen production in rat lung fibroblasts (59). Case studieshave shown that interstitial lung disease might be linked to exposure tometal dust (60, 61). Two negative results have also been reported inwhich the treatment of rats or mice with the iron chelator deferoxaminedid not inhibit bleomycin-induced lung fibrosis (62, 63). This lack ofeffects might have been due to the short period of treatment (62) or, inthe long-term study (60 days), with the single dose of bleomycintreatment (63), because single dose bleomycin-induced fibrosis normallyresolves spontaneously within 4 weeks of treatment.

This Example proposes that FENDRR inhibits fibroblast activation byreducing iron levels via interacting with IRP1, which was supported bythe following observations: (i) IRP1 was identified as the targetprotein of FENDRR by RNA pulldown-coupled mass spectrometry analysis;(ii) FENDRR overexpression inhibited ACO1 activity, reduced cellulariron levels, and decreased TFRC mRNA expression; and (iii) iron wasrequired for TGFβ-induced fibroblast activation.

How iron regulates lung fibroblast activation is still unclear and needsfurther studies. A few studies show that iron activates TGFβ signaling.One study shows that iron activates TGFβ signaling by increasing TGFβRII receptor and the phosphorylation of Smad2 in murine hepatic stellatecells (64). Another study reports that iron chelators inhibit theTGFβ/Smad pathway in prostate and colon cancer cells via the reductionin Smad2 expression and Smad3 phosphorylation due to an increase inN-myc downstream-regulated gene-1 (NDRG1) expression (65). Thus, it ispossible that FENDRR reduces iron levels, which in turn inhibits TGFβsignaling.

This Example also demonstrated another new mechanism for FENDRR activityregarding its anti-fibrotic effects, i.e., suppressing pro-fibroticmiR-214 activity by acting as its ceRNA. A growing body of evidenceindicates that IncRNAs can act as ceRNAs to sponge microRNAs (66). Aprevious publication also indicated that IncRNAs function in pulmonaryfibrosis by interacting with microRNAs to control fibroblastproliferation and activation (67). Here, it is proposed that FENDRRinhibits fibroblast activation by sponging miR-214. This is supported bythe following observations: (i) miR-214 enhanced fibroblast activation;(ii) FENDRR contains six binding sites for miR-214; (iii) FENDRRoverexpression increased the activity of a miR-214 sensor; and (iv)FENDRR directly competed with miR-214 to affect miR-214 sensor activity.However, a rescue experiment is desired to address if miR-214 canreverse the anti-fibrotic activity of FENDRR. Furthermore, the relativecontributions of iron-IRP1 and miR-214 to FENDRR activities remain to bedetermined. However, iron depletion reduces miR-214 expression,indicating that these two pathways may cross-talk.

In summary, this Example demonstrated that the IncRNA FENDRR is anantifibrotic IncRNA in the lung. This Example further demonstrated thatregulating cellular iron levels and competing with miR-214 are two novelmechanisms underlying FENDRR activity in pulmonary fibroblasts.

Thus, in accordance with the present disclosure, there have beenprovided compositions, and kits containing same, as well as methods ofproducing and using same, which fully satisfy the objectives andadvantages set forth hereinabove. Although the present disclosure hasbeen described in conjunction with the specific drawings,experimentation, results, and language set forth hereinabove, it isevident that many alternatives, modifications, and variations will beapparent to those skilled in the art. Accordingly, it is intended toembrace all such alternatives, modifications, and variations that fallwithin the spirit and broad scope of the present disclosure.

What is claimed is:
 1. An anti-fibrotic composition, comprising: anisolated and/or purified Fetal-lethal noncoding developmental regulatoryRNA (FENDRR) IncRNA or a fragment or variant thereof; and apharmaceutically-acceptable carrier.
 2. The anti-fibrotic composition ofclaim 1, wherein the FENDRR IncRNA or fragment or variant thereof isencoded by a sequence comprising at least about 100 contiguousnucleotides of at least one of SEQ ID NOS:1-3 and 133-134, or a sequencethat differs from at least one of SEQ ID NOS:1-3 and 133-134 by lessthan about 30 amino acids.
 3. The anti-fibrotic composition of claim 1,wherein the FENDRR IncRNA or fragment or variant thereof isencapsulated.
 4. The anti-fibrotic composition of claim 3, furthercomprising a delivery vehicle in which the FENDRR IncRNA or fragment orvariant thereof is encapsulated, wherein the delivery vehicle isselected from the group consisting of a liposome, a lipoplex, amicrovesicle, an exosome, a lipidoid nanoparticle, a polymericnanoparticle, an inorganic nanoparticle, and a stable nucleic acidparticle (SNALP).
 5. A pharmaceutical composition, comprising: a vectorcomprising a sequence encoding at least about 100 contiguous nucleotidesof a Fetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNAor a fragment or variant thereof; and a pharmaceutically-acceptablecarrier.
 6. The pharmaceutical composition of claim 5, wherein thesequence comprises at least about 100 contiguous nucleotides of at leastone of SEQ ID NOS:1-3 and 133-134, or a sequence that differs from atleast one of SEQ ID NOS:1-3 and 133-134 by less than about 30 aminoacids.
 7. The pharmaceutical composition of claim 5, wherein the vectoris an adenoviral vector, an adeno-associated viral (AAV) vector, analpha viral vector, a herpes viral vector, a lentiviral vector, ameasles viral vector, a pox viral vector, a phage vector, or aretroviral vector.
 8. The pharmaceutical composition of claim 5, whereinthe vector further comprises an expression control sequence to which thesequence is operably linked.
 9. A method of inhibiting activation oflung fibroblasts, the method comprising the step of: contacting the lungfibroblasts with a composition selected from the group consisting of:(i) an isolated and/or purified Fetal-lethal noncoding developmentalregulatory RNA (FENDRR) IncRNA or a fragment or variant thereof; or (ii)a vector comprising a sequence encoding at least about 100 contiguousnucleotides of a Fetal-lethal noncoding developmental regulatory RNA(FENDRR) IncRNA or a fragment or variant thereof.
 10. The method ofclaim 9, wherein in (i), the FENDRR IncRNA or fragment or variantthereof is encoded by a sequence comprising at least about 100contiguous nucleotides of at least one of SEQ ID NOS:1-3 and 133-134, ora sequence that differs from at least one of SEQ ID NOS:1-3 and 133-134by less than about 30 amino acids, and wherein in (ii), the sequencecomprises at least about 100 contiguous nucleotides of at least one ofSEQ ID NOS:1-3 and 133-134, or a sequence that differs from at least oneof SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids.
 11. Themethod of claim 9, wherein in (i), the FENDRR IncRNA or fragment orvariant thereof is encapsulated in a delivery vehicle, wherein thedelivery vehicle is selected from the group consisting of a liposome,lipid nanoparticles, a lipoplex, a microvesicle, an exosome, a lipidoidnanoparticle, a polymeric nanoparticle, an inorganic nanoparticle, and astable nucleic acid particle (SNALP).
 12. The method of claim 9, whereinin (ii), the vector is an adenoviral vector, an adeno-associated viral(AAV) vector, an alpha viral vector, a herpes viral vector, a lentiviralvector, a measles viral vector, a pox viral vector, a phage vector, or aretroviral vector.
 13. The method of claim 9, wherein in (ii), thevector further comprises an expression control sequence to which thesequence is operably linked.
 14. A method of treating or reducing theoccurrence of pulmonary fibrosis in a subject, comprising the step of:administering a composition to the subject, wherein the composition isselected from the group consisting of: (i) a composition comprising aFetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNA or afragment or variant thereof and a pharmaceutically-acceptable carrier;or (ii) a composition comprising a vector and apharmaceutically-acceptable carrier, wherein the vector comprises asequence encoding at least about 100 contiguous nucleotides of aFetal-lethal noncoding developmental regulatory RNA (FENDRR) IncRNA or afragment or variant thereof.
 15. The method of claim 14, wherein thecomposition is administered via a route selected from the groupconsisting of oral, topical, transdermal, parenteral, subcutaneous,intranasal, intratracheal, intrabronchial, mucosal, intramuscular,intraperitoneal, intravitreal, and intravenous routes.
 16. The method ofclaim 14, wherein in (i), the FENDRR IncRNA or fragment or variantthereof is encoded by a sequence comprising at least about 100contiguous nucleotides of at least one of SEQ ID NOS:1-3 and 133-134, ora sequence that differs from at least one of SEQ ID NOS:1-3 and 133-134by less than about 30 amino acids, and wherein in (ii), the sequencecomprises at least about 100 contiguous nucleotides of at least one ofSEQ ID NOS:1-3 and 133-134, or a sequence that differs from at least oneof SEQ ID NOS:1-3 and 133-134 by less than about 30 amino acids.
 17. Themethod of claim 14, wherein in (i), the FENDRR IncRNA or fragment orvariant thereof is encapsulated in a delivery vehicle, wherein thedelivery vehicle is selected from the group consisting of a liposome, alipoplex, a microvesicle, an exosome, a lipidoid nanoparticle, apolymeric nanoparticle, an inorganic nanoparticle, and a stable nucleicacid particle (SNALP).
 18. The method of claim 14, wherein in (ii), thevector is an adenoviral vector, an adeno-associated viral (AAV) vector,an alpha viral vector, a herpes viral vector, a lentiviral vector, ameasles viral vector, a pox viral vector, a phage vector, or aretroviral vector.
 19. The method of claim 14, wherein in (ii), thevector further comprises an expression control sequence to which thesequence is operably linked.
 20. The method of claim 14, further definedas a method of treating or reducing the occurrence of idiopathicpulmonary fibrosis in a subject.